Importance of NK Cells in Cellular and Humoral Responses Triggered by Pneumococcus Vaccination

Abstract

Introduction: Despite the success of vaccination in reducing overall rate of pneumococcal pneumonia, Streptococcus pneumoniae is still held responsible for high mortality and modality rates worldwide. Our study aimed to investigate the potential role played by NK cells in immune response generated by pneumococcal vaccination, which could contribute to the development of more effective vaccines.

Methods: The study included mice with and without NK cell depletion which were immunized with pneumococcus polysaccharide-conjugated vaccine followed by pneumococcus polysaccharide vaccine (PPV). Serum samples and splenocytes were collected from mice sacrificed 4 weeks after the last PPV dose. Serum samples were used for antibody level quantification by ELISA assay, while splenocytes were treated with PPV in vitro before monitoring CD4+ T-cell subsets (TH1, TH2, and TH17) and cytokine (IFN-γ, IL-4, and IL-17) secretion levels by flow cytometry and ELISA analysis, respectively.

Results: Results demonstrated reduced pneumococcal IgG and TH1 cell levels due to NK cell depletion. Nevertheless, in contrast to these observations, IFN-γ secretion levels after in vitro PPV-23 treatment of splenocytes did not exhibit any statistically significant difference between the two mice groups.

Conclusions: The data indicate a positive contribution of NK cells to both T-cell and B-cell responses triggered against pneumococcal vaccination. Further studies are required to confirm our data and investigate the potential benefit of NK cell targeting in promoting vaccine efficacy, especially in the elderly population who continues to be affected significantly by pneumococcal pneumonia.

Keywords: IgG; NK cells; Streptococcus pneumoniae; TH1; Vaccination.

Fig. 1.

Confirmation of NK cell depletion upon administration of anti-mouse NK 1.1 antibody. NK cell levels in the blood samples collected before the injection of the first anti-mouse NK1.1 antibody dose (D = 0) were compared with those obtained from the same mice one day prior to vaccination with PCV-13 (D = 5) and PPV-23 (D = 33) dose. Blood samples collected from facial veins were treated with RBC Lysis buffer and labeled with anti-mouse NK1.1-PE before flow cytometry analysis. The percentages of NK cells displayed a significant reduction 1 day prior to vaccination with PCV-23 (D = 5) and PPV-13 (D = 33) as a result of treatment with antibodies against mouse NK1.1.

Fig. 2.

Gating strategy to identify T_H populations of interest. Lymphocyte populations were gated based on FSC/SSC, excluding aggregates. T_H cells were then gated from CD3+CD4+ cells, and T_H1, T_H2, and T_H17 cell populations were identified based on IFN-γ, IL-4, and IL-17 positivity, respectively.

Fig. 3.

Comparison of PPV-23-specific IgG antibody levels. Serum samples collected 4 weeks after PPV-23 vaccination were used in ELISA analysis for evaluation of pneumococcal IgG levels. Significantly lower OD values were obtained from mice with NK cell depletion (NK−), when compared with that from control animals without NK cell depletion (NK+). ** represents p < 0.01.

Fig. 4.

Comparison of CD4+ T-cell subset levels. Splenocytes collected 4 weeks after the PPV-23 vaccination were stimulated by PPV-23 vaccine antigen for 24 h. Antibodies against IFN-γ, IL-4, and IL-17 were utilized to detect the frequencies of T_H1 (a), T_H2 (b), and T_H17 (c) cells among CD4+ T-cell population, respectively, by flow cytometry analysis. While T_H1 percentages were reduced, T_H2 and T_H17 cell ratios stayed unaltered in NK cell-depleted (NK−) mice in comparison to the control mice (NK+). ** represents p < 0.01.

Fig. 5.

Comparison of cytokine secretion levels. Splenocytes obtained 4 weeks after the last dose of combined pneumococcal vaccination were stimulated with PPV-23 vaccine antigen in vitro, and secretion levels of IFN-γ (a), IL-4 (b), and IL-17 (c) cytokines were quantified by ELISA assay of the supernatants collected. None of the cytokines displayed statistically significant difference in secretion levels between control (NK+) and NK cell-depleted (NK−) mice.